Fluid Addition To Immiscible Fluid Discrete Volumes Using An Electric Field

ABSTRACT

Compositions are provided comprising a first fluid, in the form of discrete volumes, wherein the discrete volumes are spaced apart from one another by a second fluid that is immiscible with the first fluid. According to various embodiments, the composition can comprise a surface active agent, for example, a surfactant, that lowers, minimizes, and/or eliminates coalescence between two adjacent discrete volumes of the first fluid, in the absence of an applied electric potential or field. In some embodiments, the first fluid can comprise an aqueous solution, for example, containing biological material and/or reagents, and the second fluid can comprise an oil. Systems are also provided that can comprise a conduit system for flowing compositions such as those described above, and circuitry configured to supply an electric potential and/or electric field at, near, and/or adjacent, a junction in the conduit system. The electrical potential, and/or electric field generated thereby, can be matched to the coalescence properties of the composition such that, when applied, an addition fluid continuously flowed into a junction with the composition can be added directly to a discrete volume of the composition as it passes through the junction. Methods using the compositions and systems are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 12/036,069 filed Feb. 22, 2008, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 60/891,225, and 60/891,211, which were filed on Feb. 22, 2007, and are explicitly incorporated in their entireties by reference herein.

FIELD

The present teachings relate to methods of fluid manipulation and systems and compositions for carrying out such methods.

BACKGROUND

Discrete volumes of a first fluid separated from one another by a second fluid that is immiscible with the first fluid, can sometimes undesirably coalesce with one another. Efforts to prevent such coalescence of discrete volumes can render the addition of a miscible fluid to the discrete volumes unfeasible. It would be desirable to prevent coalescence between two such adjacent immiscible fluid discrete volumes yet permit the addition of another miscible fluid into one or both of the adjacent discrete volumes.

SUMMARY

The present teachings relate to compositions of a first fluid, in the form of discrete volumes, wherein the discrete volumes are spaced apart from one another by a second fluid that is immiscible with the first fluid. According to various embodiments, the composition can comprise a surface active agent, for example, a surfactant, that lowers, minimizes, and/or eliminates coalescence between two adjacent discrete volumes of the first fluid in the absence of an applied electric potential or field. In some embodiments, the first fluid can comprise an aqueous solution, for example, containing biological material and/or reagents, and the second fluid can comprise an oil.

According to various embodiments, a system can be provided that can comprise a conduit system for flowing compositions such as those described above, and circuitry configured to supply an electric potential and/or electric field at, near, and/or adjacent, a junction in the conduit system. The electrical potential, and/or electric field generated thereby, can be matched to the coalescence properties of the composition such that, when applied, a predetermined volume of a body of addition fluid being introduced into the junction through another conduit of the conduit system can be made to flow directly into the discrete volume of the first fluid, thereby making a larger discrete volume of first and addition fluid. All other things being constant, if the electric potential, and/or electric field, generated thereby are absent, the volume of addition fluid introduced to the junction will form a separate, discrete volume of addition fluid near or next to the discrete volume of the first fluid, without coalescing, as the composition flows through the junction.

According to various embodiments, a method is provided whereby miscible fluid discrete volumes of a first fluid, separated from one another by a second fluid that is immiscible with the first fluid, can be flowed through a conduit and to a junction. A third fluid that is miscible with the first fluid can be added to a discrete volume of the first fluid at the junction. According to various embodiments, the method can comprise applying an electric potential and/or field at or near the junction to facilitate coalesceability of the miscible addition fluid with a first fluid discrete volume at the junction. In some embodiments, the junction can comprise an intersection between a first conduit and an addition-fluid supply conduit. The addition fluid can be supplied to the junction through the addition-fluid supply conduit. The addition fluid provided at the junction can separate from the body of addition fluid in the addition-fluid supply conduit and join, or in other words, coalesce with one or more first fluid discrete volumes at the junction, without forming a separate discrete volume as an intermediate step.

According to various embodiments, a method can be provided whereby a minimum electrical potential can be matched to a composition comprising a surfactant such that a discrete volume of the first fluid in the composition does not coalesce with another miscible fluid present at a junction in the absence of the electrical potential but does coalesce with a volume of a miscible addition fluid when at least the minimum electrical potential is applied.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. In the drawings:

FIG. 1 is a cross-sectional view of a system for carrying out a single molecule workflow according to various embodiments;

FIG. 2 is a cross-sectional view of a system for carrying out a variable input workflow according to various embodiments;

FIGS. 3A and 3B are cross-sectional close-up views of a system comprising a junction, and electrical circuitry for providing an electric field at the junction;

FIGS. 4 and 5 are schematic diagrams of two respective processing systems according to various embodiments;

FIGS. 6-8 depict the formulae of three different surface active agents that can be used in compositions according to various embodiments;

FIG. 9 is a cross-sectional close-up view of a system comprising a junction, and electrical circuitry for providing an electric field at the junction;

FIG. 10 is a cross-sectional close-up view of a system comprising a junction, and electrical circuitry for providing an electric potential near the junction; and

FIGS. 11-15 depict various systems and methods according to various embodiments for five respective different processing methods.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

According to various embodiments, a composition is provided that comprises an oil, for example, a non-fluorinated polyalkylpolysiloxane oil, discrete volumes of an aqueous fluid in the oil, and a surface active agent. The oil can comprise a silicone oil, for example, a non-fluorinated polyalkylpolysiloxane oil. The discrete volumes of aqueous fluid can be immiscible with one another and the discrete volumes can be spaced apart from one another with the oil in between. The composition can be provided in a conduit having an inner diameter that is about equal to a diameter of the discrete volumes of aqueous fluid, such that the discrete volumes can be arranged in a single file from within the conduit.

The surface active agent can be soluble in the oil and can have a hydrophilic/lipophilic balance (HLB) of from about 1.0 to about 8.0, from about 3.0 to about 6.0, for example, or from about 4.0 to about 5.0.

According to various embodiments, the surface active agent can be present in the non-fluorinated, polyalkylpolysiloxane oil at a concentration sufficient to prevent coalescence of the discrete volumes with one another in the absence of an applied electric potential for a period of 48 hours, when kept at a temperature in the range of about 0° C. to about 40° C. In some embodiments, the surface active agent can be present in the non-fluorinated, polyalkylpolysiloxane oil at a concentration sufficient to prevent coalescence of the discrete volumes with one another in the absence of an applied electric potential up to a temperature of at least 95° C. In some embodiments, the surface active agent can have an HLB and be present in the non-fluorinated, polysiloxane oil at a concentration that does not prevent coalescence of a discrete volume with a proximate body of addition fluid that is miscible with the first fluid of the discrete volume in the presence of an applied electric potential, for example, an applied electric potential of from about 5.0 volts to about 20 volts, AC, at about 0.1 kHz frequency or higher. The voltage can be applied at any suitable frequency, for example, at a frequency of at least about 1.0 kHz, at a frequency of from about 1.0 kHz to about 100 kHz, at a frequency higher than 100 kHz, or at a frequency of at least about 4.0 kHz. In some embodiments, a balance between HLB and concentration can be used to formulate a composition that maintains discrete volumes of a fluid, for example, discrete volumes of an aqueous fluid, that are in proximity to each other, separate and distinct from each other without coalescing, at least in the absence of a sufficiently large applied electric field, wherein the discrete volumes of such composition do coalesce with each other when in close proximity, upon application of an appropriate electric field at the interfaces thereof.

According to various embodiments, the oil can comprise a siloxane chain or ring according to the following structural formulas:

where R1, R2 can each an alkyl, aryl, vinyl, or trifluoropropyl; R3 can be an alkyl, hydroxyl, or acetyl; and n is within the range of 2-40 and m is within the range of 3-6.

According to various embodiments, the oil can comprise a polysiloxane oil and the polysiloxane oil can comprise a di-, tri-, tetra-, or penta-, siloxane. In some embodiments, the polysiloxane oil can comprise a blend or mixture of two or more different oils. In some embodiments, the polysiloxane oil can comprise a blend or mixture of two or more polysiloxane oils. According to various embodiments, the polysiloxane oil can have a viscosity ranging from about 0.25 centistoke (cSt) to about 5.0 cSt, or from about 0.50 cSt to about 3.0 cSt, for example, from about 0.65 cSt to about 2.0 cSt.

According to various embodiments, the oil can comprise a polysiloxane oil and the polysiloxane oil can comprise a polydialkylcyclosiloxane or a blend of oils that comprises an polydialkylcyclosiloxane. According to various embodiments, the polydialkylcyclosiloxane can comprise a polydimethylcyclosiloxane. According to various embodiments, the polydimethylcyclosiloxane can comprise a decamethylcyclopentasiloxane.

According to various embodiments, the polysiloxane oil can comprise polyalkylpolysiloxane. According to various embodiments, the polyalkylpolysiloxane can comprise poly-methyl-, propyl-, butyl-, pentyl-, hexyl-, octyl-, decyl-, polysiloxane. According to various embodiments, the dimethylpolysiloxane can comprise a methyl di-, tri-, tetra-, pent-, hexa-, octa-, deca-, siloxane.

In some embodiments, the composition can comprise a polyalkylpolysiloxane oil having a viscosity of from about 0.25 centistoke to about 3.0 centistokes at 25° C., for example, from about 0.5 centistoke to about 2.0 centistokes at 25° C. In some embodiments, for example, an oil can be used that also exhibits a viscosity in one or more of such ranges, at 90° C. The non-fluorinated, polyalkylpolysiloxane oil can, in some embodiments, comprise one or more of an alkyldisiloxane oil, an alkyltrisiloxane oil, and an alkyltetrasiloxane oil, and the alkyl groups can comprise methyl, ethyl, propyl, and the like, alkyl groups. In some embodiments, the oil can comprise a polydimethylpolysiloxane such as decamethyltetrasiloxane. In some embodiments, the oil can be non-fluorinated.

In some embodiments, the oil can comprise a mixture or blend of two or more oils, for example, a mixture of a polyalkylpolysiloxane oil and a polycyclosiloxane oil. An exemplary mixture comprises a ten to one weight ratio of decamethyltetrasiloxane to decamethylpentacyclosiloxane.

According to various embodiments, the surface active agent can be provided as a polydialkylsiloxane-polyalkyleneoxide at a concentration of from about 1.0% to about 20.0% by weight, dispersed in an alkylcyclosiloxane. According to various embodiments, the surface active agent can comprise about one percent by weight solution of alkylsiloxane-polyalkyleneoxide dispersed in a polysiloxane oil, which is then mixed with oil to form the first fluid described herein. In some embodiments, the surface active agent can comprise, for example, a detergent, a wetting agent, or an emulsifier.

According to various embodiments, the surface active agent can comprise a polyalkylene oxide. According to various embodiments, the polyalkylene oxide can comprise a polyethylene oxide (PEO) such as polyethylene glycol (PEG) or polypropylene glycol (PPG).

According to various embodiments, the surface active agent can comprise a backbone of polysiloxane, substituted with a polyalkylene oxide. According to various embodiments, the polysiloxane backbone can comprise a polyalkylsiloxane cross-linked with a polyalkylene oxide that can form a gel particle of average diameter ranging from about 1 micron to about 10 micron. According to various embodiments, the average diameter of the gel particle is about 5 microns.

According to various embodiments, the surface active agent can comprise a polyalkyleneoxide-substituted siloxane, for example, a polyethylene glycol-substituted siloxane. An exemplary surface active agent is DC 9011 surfactant from Dow Corning. The surface active agent can be dispersed in a carrier oil before it is mixed with the oil described herein. For example, the surface active agent can be dispersed in a polysiloxane oil or in a polycyclosiloxane oil, before it is mixed with a polyalkylpolysiloxane oil. In an exemplary embodiment, the surface active agent can comprise a polyalkyleneoxide-substituted polysiloxane dispersed in a cyclic polysiloxane oil, for example, in D5, a decamethylpentacyclosiloxane oil.

According to various embodiments, the surface active agent can comprise a silicone polyether, for example, PEG/PPG-18 dimethicone (10% by weight) in cyclopentasiloxane. An exemplary surfactant of this type is SILSURF 400 R available from Siltech Corporation of Toronto, Ontario, Canada. Like other surface active agents, SILSURF 400R can be provided as a dispersion in a carrier oil, such as a polycyclosiloxane oil or decamethylpentacyclosiloxane, for example, in a 10% by weight dispersion, based on the total weight of the dispersion. The carrier oil can be miscible with the oil used as the second fluid, and can make up from about 1% to about 30% by weight of the total weight of the oil, carrier oil, and surface active agent, combined, for example, 10% by weight of such total weight.

Another exemplary surface active agent that can be used is GRANSURF 77 available from Grant Industries, Elmwood Park, N.J. GRANSURF 77 comprises PEG-10 dimethicone and can be provided neat or in a dispersion, for example, in a dispersion comprising a polycyclosiloxane oil.

The surface active agent can be provided in any suitable concentration in an oil or oil mixture. Exemplary concentrations can include, for example, about 0.1 to about 10% by weight surface active agent based on the total weight of the combined surface active agent and oil or oil blend. Concentrations of from about 0.25% by weight to about 5.0% by weight can be used, as can concentrations of from about 0.5% by weight to about 3.0% by weight, for example, about 1.0% by weight surface active agent. In some embodiments, greater concentrations of surface active agent can be used with higher applied electric potentials, relative to the electric potential that can be used with lower concentrations of surface active agent.

According to various embodiments, the oil can comprise a fluorinated oil and the surface active agent can comprise a compound soluble in the fluorinated oil at a concentration sufficient to prevent discrete volumes of aqueous fluid therein from coalescing in the absence of an applied electric field.

According to various embodiments, a system is provided that comprises: a first conduit; a composition as described above, in the first conduit; a second conduit in fluid communication with the first conduit at a junction; and electric circuitry configured to apply an electric potential and/or field at the junction. An addition fluid can be present in the second conduit, and the addition fluid can be miscible with the aqueous fluid and non-coalesceable with the discrete volumes in the absence of the electric potential. The electric circuitry can be configured to apply an electric potential of at least about 1.0 volt, for example, at least about 5.0 volts, alternating current, at the junction. The voltage can be applied at a frequency of at least about 0.1 kHz, for example, at least about 1.0 kHz. An exemplary frequency can be, for example, at least about 4.0 kHz. In an exemplary embodiment, the electric circuitry can be configured to apply an electric potential of from about 5.0 volts to about 20 volts alternating current at a frequency of from about 4.0 kHz to about 50 kHz, to form an electric field at the junction.

The electric circuitry can comprise an electrode that contacts the second conduit, for example, an electrode sleeve surrounding a portion of the second conduit at the junction. The first conduit can comprise a portion upstream of the junction and a portion downstream of the junction, and the electric circuitry can comprise at least a first electrode disposed along the portion of the first conduit upstream of the junction, downstream of the junction, and/or at the junction. The electric circuitry can also comprise at least a second electrode disposed along the second conduit. In some embodiments, each of the first conduit and the second conduit can comprise an outer surface comprising an electrically insulating material, for example, a plastic material. In some embodiments, the first electrode can be in contact with the outer surface of the first conduit while the second electrode can be in contact with the outer surface of the second conduit. In some embodiments, the conduits can comprise a polyolefin, such as polyethylene or polypropylene, a fluoropolymer such as polytetrafluoroethylene (PTFE), or a polyethyleneterephthalate (PET). Other materials that can be used for the conduits include perfluoroalkoxy-2 (PFA), fluorinated ethylene propylene (FEP), polyvinylidene fluoride (PVDF, e.g., KYNAR™), and ethylenetetrafluoroethylene (ETFE, e.g., TEFZEL™). In various embodiments, the fluidic pathways can be present between layers, such as those milled or molded on a chip.

According to various embodiments, the system can comprise a pump configured to move a composition including immiscible fluid discrete volumes along the first conduit from upstream of the junction to downstream of the junction. In some embodiments, the pump can cause a negative pressure from a downstream side of the junction to pull fluids through the first conduit, and in some embodiments, the pump can cause a positive pressure from an upstream side of the junction to push fluids through the first conduit. Positive pressures of from about 2 psig to about 20 psig, for example, from about 8 psig to about 12 psig, can be used according to various embodiments. In some embodiments, pressure is used to reduce expansion of aqueous discrete volumes during heating, for example, during heating to temperatures needed for polymerase chain reaction, and in some embodiments, to temperatures from about 94° C. to about 96° C.

According to various embodiments, a system is provided that comprises: a first conduit comprising an outer surface that comprises an electrically insulating material; a second conduit in fluid communication with the first conduit, at a junction, the second conduit comprising an outer surface that comprises an electrically insulating material, wherein the first conduit comprises a portion upstream of the junction and a portion downstream of the junction; and electric circuitry comprising at least a first electrode disposed along the first conduit and at least a second electrode disposed along the second conduit. Although the portions of the first conduit upstream and downstream of the junction can comprise separate, different conduits, the portions are nonetheless together referred to herein as portions of the first conduit. In some embodiments the first electrode can be disposed along the portion of the first conduit upstream of the junction. In some embodiments the first electrode can be disposed along the portion of the first conduit downstream of the junction. In some embodiments the first electrode can be disposed along the first conduit at the junction. According to various embodiments, either or both conduits can comprise an electrically conductive material can be used, for example, a carbon-filled plastic material.

In some embodiments, the first electrode can be in contact with the outer surface of the first conduit, and the second electrode can be in contact with the outer surface of the second conduit. The electrodes do not need to penetrate the conduits and contact liquid inside the conduits but can instead be clamped to, or otherwise in contact with, the outer surface of each conduit. In some embodiments the electric circuitry can comprise an electrode sleeve surrounding a portion of the second conduit at the junction. In some embodiments, the electric circuitry can be configured to apply an electric potential of from about 1.0 volt to about 100 volts alternating current, for example from about 5.0 volts to about 20 volts. The voltage can be applied, for example, at a frequency of from about 1.0 kHz to about 100.0 kHz, for example, at from about 5 kHz to about 50 kHz, or at, at least about 4 kHz.

In some embodiments, the electric circuitry can be configured to apply an electric potential of from about 5.0 volts to about 20.0 volts alternating current, for example, at a frequency of at least about 1.0 kHz. The electric circuitry can apply alternating current of any of a variety of waveforms, for example, the alternating current can be in the form of a sine wave, a square wave, a pulsed wave, a ramp wave, an arbitrary wave, a saw-tooth wave, or the like.

According to various embodiments, a method of fluid addition is provided whereby an addition fluid can be made to coalesce with a discrete volume of a first fluid. The method can comprise: flowing a composition of fluids through a first conduit, the composition of fluids comprising discrete volumes of a first fluid in a second fluid. The first fluid and the second fluid can be immiscible with one another and the discrete volumes can be spaced apart from one another by the second fluid. The method can comprise positioning a first of the discrete volumes at a junction along the first conduit, wherein the junction comprises an intersection of a second, or addition, conduit that is in fluid communication with the first conduit. The method can further comprise flowing a continuous supply of the third (addition) fluid, in contrast to a second composition of discrete volumes of the addition fluid in the second fluid, through the second conduit and into the junction so that a portion of the continuous supply of addition fluid contacts the first discrete volume. The third fluid can be miscible with the first fluid but non-coalesceable with the first discrete volume in the absence of an applied electric field. The method can also comprise applying an electric field to the junction to cause the portion of the continuous supply of third fluid to coalesce with the first discrete volume at the junction, yet to not cause the first discrete volume to coalesce with its adjacent discrete volumes in the composition.

The method can comprise continuously flowing the body of third fluid, which fills the addition-fluid supply conduit, into the junction at a constant rate. The method can comprise flowing the body of third fluid, which fills the addition-fluid supply conduit, periodically, intermittently, in a pulsed fashion, or in a like manner into the junction.

In some embodiments, detection of discrete volumes reaching the junction can be used to determine and/or adjust the flow of third fluid through the second conduit and to the junction.

In some embodiments, the method can process a composition of fluids that comprises a surface active agent that is soluble in the second fluid and that has a hydrophilic/lipophilic balance of from about 2 to about 8, for example, from about 3 to about 7 or from about 4 to about 5. In some embodiments where the volume of the discrete volumes before addition is within the range of about 100 nanoliters to about 1000 nanoliters, the surface active agent used according to the method can be present in the second fluid at a concentration of from about 0.1% by weight to about 10% by weight, based on the total weight of the first second and surface active agent. Discrete volumes having volumes in the 10 picoliter to 10s of nanoliters may use a lower concentration of surface active agent to prevent coalescence with each other in the absence of an applied electric field. The surface active agent can, for example, have a hydrophilic/lipophilic balance that does not prevent coalescence of the discrete volume with an addition fluid in the presence of an applied electric potential yet which does prevent coalescence in the absence of an applied electric potential. An exemplary applied electric potential, that can be used, for example, as an exemplary threshold value, can be at least about 5.0 volts alternating current at a frequency of at least about 1.0 kHz, for example, 7.5 volts square wave alternating current at a frequency of 4 kHz.

The method can involve the use of a first fluid that comprises an aqueous fluid. The method can involve the use of a second fluid that comprises a non-fluorinated polyalkylpolysiloxane oil. In some embodiments, the third fluid, which is to be added to selected discrete volumes of the first fluid, can comprise an aqueous fluid, for example, aqueous-based polymerase chain reaction reagent or reagent mixture. In some embodiments, the third fluid can comprise an aqueous-based sequencing reaction reagent or reagent mixture, for example, a mixture of reagents to carry out a forward or reverse Sanger cycle sequencing reaction. In some embodiments, the third fluid can comprise aqueous-based PCR amplification clean-up compounds, e.g., exo-SAP. In some embodiments, the method involves the use of a surface active agent that comprises a polyalkyleneoxide-substituted siloxane, for example, a polyethylene glycol-substituted siloxane. In some embodiments, the first fluid can comprise a non-fluorinated polyalkylpolysiloxane oil, and the surface active agent can comprise a polyalkyleneoxide-substituted polysiloxane. According to various embodiments of the method, the first fluid can comprise an oil, the second fluid can comprise an aqueous fluid, and the surface active agent can be one that exhibits a hydrophilic/lipophilic balance of from about 1 to about 7, or from about 3 to about 6, or from about 4 to about 5.

In some embodiments, the method can comprise applying an electric potential of at least about 1.0 volt, alternating current, at the junction, for example, at least about 5.0 volts. The method can comprise applying voltage at a frequency of at least about 1.0 kHz, at the junction, for example, at a frequency of at least about 4 kHz, or from about 1.0 kHz to about 100 kHz.

In some embodiments, electric circuitry can be provided that generates an electric field encompassing the junction. The electric field can be generated by applying an electric potential of at least about 1.0 volt alternating current (AC). Exemplary potentials comprise the ranges of from about 1.0 volt to about 100 volts, from about 5.0 volts to about 50 volts, and from about 15 volts to about 20 volts, for a system comprising, for example, polytetrafluoroethylene conduits having a 0.035 inch outer diameters and 0.024 inch inner diameters, and a composition that comprises discrete volumes flowing through the conduits each having a diameter that about equals the inner diameters of the conduits. Such exemplary potentials can be applied by placing the two electrodes each about two inches away from the junction, for example, the first electrode contacting the first conduit downstream of the junction, and the second electrode contacting the second or addition conduit. Other spacings away from the junction can be used instead, for example, spacings of from about 0.1 inch to about 10 inches away from the junction can be used to position the electrodes relative to the junction. The spacing can be in any radial direction away from the junction, for example, in directions along the respective conduits. In some embodiments, a first electrode contacts the addition conduit along the addition conduit and about one to five inches away from the junction, and another electrode floats, in effect being coupled to the first conduit through the capacitive effects of the air, etc. Other diameters for the conduits can also be used, for example, conduits having from about 0.001 to about 0.2 inch inner and outer diameters, or from about 0.005 inch to about 0.1 inch inner diameters and from about 0.010 inch to about 0.015 inch outer diameters.

According to various embodiments, the discrete volumes traveling through the first conduit, before and/or after a junction along the first conduit, can be oblong in shape and can have, for example, a cross-sectional shape that generally matches and can be defined by the cross-sectional shape of the inside of the first conduit. For example, in embodiments where the first conduit has a circular inner cross-section, each discrete volume traveling through the first conduit likewise has a circular cross-section of just slightly smaller dimension than the dimension of the conduit inner cross-section. While the diameter of each discrete volume will always be less than the inner diameter of the first conduit through which the discrete volume is moving, the diameter of each discrete volume can be more than 75% of the inner diameter of the first conduit, for example, greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the inner diameter of the first conduit. In some embodiments, each discrete volume can have a cross-sectional diameter that is from about 95% to about 99.9% of the inner diameter of the conduit in which the discrete volumes are disposed. In some embodiments, the shape of each discrete volume in the first conduit can be oblong such that each discrete volume has an axial dimension. The leading and trailing faces of each discrete volume can comprise curved surfaces, for example, hemispherical surfaces, and a middle portion of each discrete volume can have a cylindrical shape when traveling through a straight portion of the first conduit. According to various embodiments, the axial dimension of each discrete volume can be less than or greater than the cross-sectional dimension thereof. In some embodiments, when greater, the axial dimension of each discrete volume can be from about 110% to about 1000% the cross-sectional diameter of each discrete volume, for example, from about 150% to about 750%, or from about 200% to about 500%. Although the discrete volumes will take-on a cross-sectional shape that roughly matches the cross-sectional shape of the first conduit through which they are traveling, surface tension in aqueous discrete volumes tend to cause the discrete volumes to take on rounded surfaces as opposed to sharp, angular corners, even when traveling through a first conduit with a square cross-section.

According to various embodiments, each discrete volume can have an axial length that is less than the diameter of the discrete volume. In such cases, the axial dimension can be measured from the front or leading face of the discrete volume to the back or trailing face of the discrete volume. The axial dimension can be collinear with the axial dimension of the first conduit. In some embodiments, the axial dimension can be from about 30% to about 100% of the diameter of each discrete volume, for example, from about 50% to about 90% or from about 60% to about 80% of the diameter.

In some embodiments, discrete volumes that are to be processed according to various embodiments of the present teachings can comprise volumes of liquid within the range of from about 100 nanoliters (nL) to about 100 microliters (μL), depending upon the inner diameter of the first conduit through which discrete volumes are made to travel. In some embodiments, for tubing having an inner diameter of 0.024 inch, the discrete volumes to be processed at a junction can enter the junction having volumes of from about 200 nL to about 2000 nL, each, for example, from about 300 nL to about 1000 nL or from about 400 nL to about 500 nL, each. In some embodiments, larger discrete volumes of, for example, from about 1000 nL to about 2000 nL can be split by introducing oil into the discrete volumes at a junction, as described herein, to provide smaller discrete volumes of from about 200 nL to about 500 nL, each, for example, of about 400 nL each.

Other combinations of temperature, voltages, frequencies, tubing material, diameters, vicinities of electrodes to the junction, and sizes and shapes of the discrete volumes, can be used to provide a system that enables the application of an electric field of sufficient strength and vicinity to cause addition fluid flowing into a junction to coalesce with a first discrete volume and yet not cause electrocoalesence of the first discrete volume and its adjacent discrete volumes.

According to various embodiments of the method, the first conduit can comprise a portion upstream of the junction and a portion downstream of the junction, and the method can comprise applying an electric potential to a first electrode disposed along the portion of the first conduit downstream of the junction. In some embodiments, the method can comprise applying potential to a second electrode disposed along the second conduit. In some embodiments, the method can comprise applying an electric potential to an electrode sleeve surrounding a portion of the second or addition conduit, at the junction. The electrode sleeve can comprise, for example, a metal sleeve or a sleeve made out of any other electrically conductive material. The sleeve can comprise copper, aluminum, stainless steel, brass, nickel, platinum, palladium, combinations thereof, and the like. The sleeve can have an inner diameter that about equals or is only slightly larger than the outer diameter of the conduit around which the sleeve is disposed. In some embodiments, the sleeve has an inner diameter that is from about 1.0 micron to about 1.0 millimeter larger than the outer diameter of the conduit around which the sleeve is disposed. In some embodiments, both the first and second electrodes can comprise a sleeve.

In some embodiments, the method can comprise applying electric potential to each of the first conduit and the second conduit, wherein each conduit comprises an outer surface comprising an electrically insulating material. In some embodiments, the entire conduit can comprise the same material. In some embodiments, the conduits can comprise plastic tubing, for example, polytetrafluoroethylene tubing. The method can comprise contacting the outer surface of the first conduit with the first electrode and contacting the second electrode with the outer surface of the second conduit.

In some embodiments, the method can comprise flowing fluids through one or more of the conduits by pushing the fluids, for example, pushing the fluids through the first conduit and through the addition conduit. The system can comprise one or more pumps and an appropriate control system for controlling flow rate, pressure, and/or other parameters that involve the movement of the composition of discrete volumes of a first fluid in a second fluid through the system. Likewise, the system can comprise one or more pumps and an appropriate control system for controlling flow rate, pressure, and/or other parameters that involve the movement of addition fluid through a second or addition conduit of the system.

The method can comprise pulling a composition of fluids through the first conduit, for example, a composition comprising discrete volumes of a first fluid in a second fluid, wherein the first and second fluids are immiscible with respect to one another. The method can further comprise pushing a third fluid through the second conduit and into the junction, for example, by intermittently, periodically, independently, or on demand, pushing a volume of the third fluid through the second conduit, through the junction, and into the first conduit. Even spacing between discrete volumes traveling through the first conduit and into the junction can be used to provide additions of equal volume to each of the discrete volumes, if the discrete volumes pass the junction at a constant rate. For example, 100 nanoliter-sized portions of the third fluid can be added to each of a plurality of 500 nanoliter-sized discrete volumes of the first fluid.

In some embodiments, the method can comprise continuously feeding or pumping addition fluid, as opposed to intermittently, through the second conduit and through the junction, into the first conduit. If continuously fed, the third (addition) fluid can be fed at a constant flow rate through the addition conduit and into the first conduit. The continuous flow can be of sufficient volume, relative to the flow of discrete volumes through the first conduit, to provide additions, each of a volume that about equals from about 1% to about 50% of the volume of a respective discrete volume to which the addition is to be made, for example, the flow rate of the addition fluid can be such that each addition is of a volume equal to about 20% of the volume of the respective discrete volume to which the addition is to be made.

In some embodiments, even spacing between discrete volumes traveling through the first conduit and into the junction can be provided, facilitating the addition of equal volumes to the discrete volumes. In some embodiments, the portions of the third fluid to be added to discrete volumes at the junction can be from about 1% by volume to about 50% by volume of the size of the discrete volumes to which the third fluid is to be added, for example, from about 5% by volume to about 40% by volume, or from about 10% to about 30% by volume. In some embodiments, about a 100 nanoliter-sized portion of third fluid can be added to a 500 nanoliter-sized discrete volume. In another example, portions of third fluid can be added to 500 nanoliter-sized discrete volumes, and the addition fluid portions can be from about 50 nanoliters to about 200 nanoliters, or from about 75 nanoliters to about 150 nanoliters.

Other methods of pushing and pulling fluids into and through a junction are described, for example, in greater detail in each of U.S. patent application Ser. No. 11/507,735, U.S. patent application Ser. No. 11/508,044, U.S. patent application Ser. No. 11/508,756 and U.S. patent application Ser. No. 11/507,733, all filed on Aug. 22, 2006, which are incorporated herein in their entireties by reference.

In some embodiments, the method can comprise applying an electric potential, for example, an electric potential of at least about 5.0 volts alternating current, for example, from about 5.0 volts to about 20 volts alternating current. The voltage can be applied at a frequency of at least about 1.0 kHz, for example, at a frequency of from about 1.0 kHz to about 100.0 kHz.

According to various embodiments, the second fluid can comprise a silicone oil, the surface active agent comprises a polyalkylene oxide-substituted polysiloxane, and the method can comprise applying an electric potential of at least about 5.0 volts alternating current at a frequency of at least about 1.0 kHz, for example, applying a potential of about 17 volts AC at a frequency of about 4.0 kHz to two polytetrafluoroethylene conduits (the first conduit of dimensions 0.035 inch outer diameter and 0.024 inch inner diameter, and the addition conduit of 381 microns inner diameter and 813 microns outer diameter), by contacting the conduits with respective electrodes each spaced two inches away from the junction.

In some embodiments, the electrodes can each independently be spaced any suitable distance away from the junction, for example, from about 0.1 inch to about 10 inches away from the junction, from about 0.5 inch to about 5.0 inches away from the junction, or from about 1.0 inch to about 3.0 inches away from the junction. One or both of the electrodes can comprise a sleeve of electrically conductive material, for example, a metal, surrounding one or both of the conduits, and the method can comprise contacting the sleeve with the lead/wire of a voltage source.

In some embodiments, the first conduit and the addition conduit can have the same or different inner diameters and the same or different outer diameters. In some embodiments, the addition conduit can have a smaller inner diameter than the inner diameter of the first conduit. In some embodiments, the inner diameter of the addition conduit can be from about 10% to about 90% as large as the inner diameter of the first conduit, for example, from about 25% to about 75% as large, or about 50% as large.

Referring now to the drawings, and according to various embodiments, a mixture of nucleic acids, for example, a mixture generated by a DNA library, can be diluted by a limiting dilution procedure such that a concentration of a single molecule of interest per a given volume of liquid can be obtained. Nucleic acids obtained by any method can be diluted to a concentration such that, for a given size of discrete volume, about one out of every five discrete volumes can contain a single molecule, prior to processing. When a sample is diluted to this extent, some discrete volumes made from the diluted sample may not have any nucleic acid molecules of interest at all, while others may have more than 1 molecule. For example, in some embodiments, about 20% of the discrete volumes can contain a single molecule of interest, although other concentrations can be used.

Greater details on obtaining such concentrations and distributions of molecules per discrete volume, and uses for such collections, can be found, for example, in each of U.S. patent application Ser. No. 11/507,735, U.S. patent application Ser. No. 11/508,044, U.S. patent application Ser. No. 11/508,756 and U.S. patent application Ser. No. 11/507,733, all filed on Aug. 22, 2006, which are incorporated herein in their entireties by reference.

According to various embodiments, as shown, for example, in FIG. 1, a single molecule workflow can be provided wherein a mixture of DNA 100, can be diluted such that at most about 40% of the immiscible-fluid-discrete-volumes produced from a sample in the process described below can comprise a single target nucleic acid sequence. In various other embodiments, less than about 37% of the immiscible-fluid-discrete-volumes produced can each comprise a single target nucleic acid sequence. In other embodiments, at least 1% or more, 5% or more, 10% or more, or 20% or more can comprise a single target nucleic acid sequence, for example, from about 5% to about 40%, or from about 10% to about 20%.

One of skill in the art can appreciate how to prepare a solution by limiting dilution. As illustrated in FIG. 1, after a sample 100 is diluted by limiting dilution, a volume of sample 102 comprising a single nucleic acid molecule can be introduced into a conduit 104, and passing a junction with an oil supply conduit, forms an aqueous immiscible-fluid-discrete-volume 106. Not all aqueous immiscible-fluid-discrete-volumes formed from the sample will necessarily contain a single nucleic acid molecule of interest there, and some, depicted as volumes 108, contain no molecules of interest. In various embodiments, about 10% or less, about 20% or less, or about 50% or less of the aqueous immiscible-fluid-discrete-volumes can have one nucleic acid molecule per given volume.

According to various embodiments shown in FIG. 2, a variable input workflow can be provided wherein a mixture of nucleic acids is supplied in a plate 200, for example, a mixture of genomic DNA or cDNA, and oligonucleotide primers. Nucleic acids obtained by any method can be provided in a conduit 202 at a concentration such that each of the immiscible-fluid-discrete-volumes 204 produced from the mixture comprise from about 10 to about 1000 copies of the genomic DNA or cDNA. In various embodiments, the discrete volumes can comprise from about 100 to about 500 copies of the genomic DNA or cDNA, and in some embodiments each discrete volume can comprise about 300 copies of genomic DNA or cDNA. In various embodiments, the nucleic acid mixture can be removed, injected, or recovered from one or more wells of plate 200.

Greater details on obtaining such concentrations and distributions of molecules per discrete volume, and uses for such collections, can be found, for example, in each of U.S. patent application Ser. No. 11/507,735, U.S. patent application Ser. No. 11/508,044, U.S. patent application Ser. No. 11/508,756 and U.S. patent application Ser. No. 11/507,733, all filed on Aug. 22, 2006, which are incorporated herein in their entireties by reference.

FIGS. 3A and 3B depict a system comprising a junction as described herein and electrical circuitry configured to apply an electric field at the junction. The circuitry comprises an electrode sleeve surrounding the addition conduit and another electrode contacting a T-intersection with which a first conduit and the addition conduit are in fluid communication.

FIGS. 4 and 5 depict various schematic diagrams of two respective systems according to various embodiments. Greater details about such systems and their components can be found, for example, in each of U.S. patent application Ser. No. 11/507,735, U.S. patent application Ser. No. 11/508,044, U.S. patent application Ser. No. 11/508,756, and U.S. patent application Ser. No. 11/507,733, all filed on Aug. 22, 2006, which are incorporated herein in their entireties by reference. In the schematic diagrams of FIGS. 4 and 5, the slugOmeters and SOM's shown can be the same as the various detectors shown in described in these referenced applications.

FIGS. 6-8 depict the formulae of three respective surface active agents that can be used in the compositions, systems, and methods described herein. In some embodiments, any of the surfactants shown in FIGS. 6-8 can be used in a concentration of from about 0.1% by weight to about 10% by weight, for example, from about 0.5% by weight to about 2.0% by weight, or about 1.0% by weight, based on the weight of the oil or oil blend in which the surfactant is added.

FIG. 9 depicts a system comprising a junction as described herein and electrical circuitry configured to apply an electric field at the junction. The circuitry comprises an electrode sleeve surrounding the addition conduit and another electrode contacting a T-intersection with which a first conduit and the addition conduit are in fluid communication.

FIG. 10 depicts a system comprising a junction as described herein and electrical circuitry configured to apply an electric potential near the junction. The circuitry comprises an electrode sleeve surrounding the addition conduit and another electrode capacitively coupled to a T-intersection with which a first conduit and the addition conduit are in fluid communication through, for example, the air.

As shown in FIG. 11, the system exemplified can comprise a first conduit 400 in fluid communication with a second conduit 402 at a junction 404. A composition comprising a non-fluorinated polyalkylpolysiloxane oil 406 and discrete volumes of aqueous fluid 408, travels through first conduit 400 in the direction indicated. The non-fluorinated polyalkylpolysiloxane oil and the aqueous fluid are immiscible with one another and discrete volumes of aqueous fluid 408 are spaced apart from one another. An addition fluid 410 in second conduit 402 can be added to a discrete volume of aqueous fluid 412 at the junction 404. The enlarged discrete volumes of aqueous fluid 414, comprising addition fluid 410, can then be made to travel through first conduit 400, remaining non-coalesced with one another. Electric circuitry such as that shown in FIGS. 3A and 3B can be used to provide an electric field at junction 404 sufficient to aid in the coalescence of addition fluid 410 to discrete volumes of aqueous fluid 408 at junction 404.

The system can further comprise a pump 420 and a control apparatus 422 configured to provide the addition fluid to the discrete volumes of aqueous fluid. Pump 420 and apparatus 422 can control the flow rate of addition fluid 410 so that a desired amount of addition fluid 410 can be added to each discrete volume of aqueous fluid 408 at junction 404.

Pump 420 and control unit 422 can be configured to pump addition 410 continuously, in contrast to intermittently, periodically, or on demand, into junction 404. Pump 420 and control unit 422 can be configured to pump addition 410 intermittently into junction 410, periodically into junction 410, or on demand into junction 410. In some embodiments, a pump and control unit can similarly be used to control the flow of non-fluorinated polyalkylpolysiloxane oil 406 and discrete volumes of aqueous fluid 408 traveling through first conduit 400 by pushing the composition in the direction indicated. In some embodiments, a pump and control unit can similarly be used to control the flow of non-fluorinated polyalkylpolysiloxane oil 406 and discrete volumes of aqueous fluid 408 traveling through first conduit 400 by pulling the composition in the direction indicated.

In the embodiment shown in FIG. 12, a first conduit 430 and a second conduit 432 are in fluid communication with each other at a junction 434. A composition comprising a non-fluorinated polyalkylpolysiloxane oil 436 and discrete volumes of aqueous fluid 438, travels in first conduit 430 in the direction indicated. As a discrete volume of aqueous fluid 442 travels reaches junction 434, a volume of aqueous fluid 440 can be removed from the discrete volume 442 through second conduit 432. The reduced discrete volumes of aqueous fluid 444 can then be made to continue to travel through first conduit 430, and remain non-coalesced.

In the embodiment shown in FIG. 12, a pump 420 applies a negative pressure to second conduit 432 causing the removal of aqueous fluid 440 through the second conduit 432.

In the embodiment shown in FIG. 13, a system is provided that comprises a first conduit 460, and a second conduit 462 in fluid communication with the first conduit 460, at a junction 464. A composition comprising a non-fluorinated polyalkylpolysiloxane oil 466 and discrete volumes of an aqueous fluid 468, travels through first conduit 460 in the indicated direction. As a discrete volume of aqueous fluid travels through first conduit 460 and through junction 464, additional oil 470 can be injected into the composition through second conduit 462. The aqueous fluid 472 and oil 470 are immiscible with one another. The discrete volume of aqueous fluid 472 can be split into two smaller discrete volumes 474 and 476. The discrete volumes of aqueous fluid 474, 476, and other previously split portions 478, remain non-coalesced as they continue to travel through the first conduit.

In the embodiment shown in FIG. 14, the system comprises a first conduit 500, and a second conduit 502 in fluid communication with first conduit 500 at junction 504. The composition comprises a non-fluorinated polyalkylpolysiloxane oil 506 and discrete volumes of an aqueous fluid 508 traveling through the first conduit in the indicated direction. Additional polyalkylpolysiloxane oil 510 is added to the oil 506 between the discrete volumes 508 at the junction 504. As a result, the system increases the spacing between adjacent discrete volumes of aqueous fluid by providing a greater amount of oil 514 in between adjacent discrete volumes. The discrete volumes are indicated as 512 downstream of junction 504. The discrete volumes of aqueous fluid 512 remain non-coalesced as they continue to travel through the first conduit 500.

In the embodiment shown in FIG. 15, the system comprises a first conduit 530, and a second conduit 532 in fluid communication with the first conduit 530 at a junction 534. A composition comprising a polyalkylpolysiloxane oil 536 and discrete volumes of an aqueous fluid 538, which are immiscible with the oil, travels through first conduit 530 in the indicated direction. At junction 534, a portion of a volume of polyalkylpolysiloxane oil 540 can be removed between the adjacent discrete volumes of aqueous fluid 538 and 542 on either side of the volume of oil 540, at junction 534. The system can accordingly be used to decrease the amount of oil 544, and thus the spacing, between adjacent discrete volumes of aqueous fluid 542 and 546. As depicted, the discrete volumes of aqueous fluid 546 remain non-coalesced as they continue to travel through the first conduit 530.

Other embodiments of the present teachings will be apparent to those skilled in the art from consideration of the present specification and practice of the present teachings disclosed herein. It is intended that the specification and examples be considered as exemplary only and not be limiting. All cited references, patents, and patent applications are incorporated in their entireties herein by reference. 

1-10. (canceled)
 11. A method of coalescing an addition fluid with a discrete volume of a second fluid, comprising: flowing a composition of fluids through a first conduit, the composition of fluids comprising discrete volumes of a first fluid in the second fluid, the first fluid and the second fluid being immiscible with one another and the discrete volumes being spaced apart from one another by the second fluid; positioning a first discrete volume of the discrete volumes in a junction in the first conduit, the junction comprising a second conduit in fluid communication with the first conduit; flowing a body of addition fluid through the second conduit and into the junction so that a portion of the body of addition fluid contacts the first discrete volume, the addition fluid being miscible with the first fluid but non-coalesceable with the first discrete volume in the absence of an applied electric field; and applying an electric field to the junction to cause the portion of the body of addition fluid to coalesce with the first discrete volume at the junction.
 12. The method of claim 11, wherein the composition of fluids comprises a surface active agent that is soluble in the second fluid and has a hydrophilic/lipophilic balance of from about 2 to about
 7. 13. The method of claim 12, wherein the surface active agent is present in the second fluid at a concentration of from about 0.1% by weight to about 10% by weight, based on the total weight of the second fluid and surface active agent.
 14. The method of claim 11, wherein the second fluid comprises a non-fluorinated polyalkylpolysiloxane oil and the first fluid comprises an aqueous fluid.
 15. The method of claim 11, wherein the surface active agent comprises a polyalkyleneoxide-substituted siloxane.
 16. The method of claim 11, wherein the surface active agent comprises a polyethylene glycol-substituted siloxane.
 17. The method of claim 11, wherein the second fluid comprises a non-fluorinated polyalkylpolysiloxane oil and the surface active agent comprises a polyalkyleneoxide-substituted polysiloxane.
 18. The method of claim 11, wherein the second fluid comprises an oil, the second fluid comprises an aqueous fluid.
 19. The method of claim 11, wherein the applying an electric field comprises applying an electric potential of at least about 5.0 volts, alternating current, at a frequency of at least about 4.0 kHz, at the junction.
 20. The method of claim 11, wherein the applying an electric field comprises applying an electric potential to an electrode sleeve surrounding a portion of the second conduit at the junction.
 21. The method of claim 11, further comprising continuously pushing the addition fluid through the second conduit and into the junction.
 22. The method of claim 11, wherein the applying an electric field comprises applying an electric potential of at least about 5.0 volts alternating current at a frequency of from about 4.0 kHz to about 50.0 kHz.
 23. The method of claim 11, wherein the first fluid comprises a silicone oil, the surface active agent comprises a polyalkylene oxide-substituted polysiloxane, and the applying an electric field comprises applying an electric potential of at least about 5.0 volts alternating current at a frequency of at least about 1.0 kHz. 